Concentrated collagen hydrogels as dermal substitutes Christophe Helary a, * , Isabelle Bataille b, c , Aicha Abed b, c , Corinne Illoul a , Annie Anglo a , Liliane Louedec b , Didier Letourneur b, c , Anne Meddahi-Pelle ´ b, c , Marie Madeleine Giraud-Guille a a CMCP - CNRS UMR7574, EPHE, University Pierre and Marie Curie, 4 place Jussieu, 75005 Paris, France b INSERM, U698, CHU Xavier Bichat, Ba ˆt. Inserm, 46 rue Henri-Huchard,75018 Paris, France c BPC, Institut Galile´e, Universite´ Paris 13, 93430 Villetaneuse, France article info Article history: Received 14 June 2009 Accepted 18 September 2009 Available online xxx Keywords: Dermal substitutes Concentrated collagen hydrogel Fibroblast growth Contraction Gelatinase A In vivo integration abstract Collagen hydrogels first appeared promising for skin repair. Unfortunately, their extensive contraction and their poor mechanical properties constituted major disadvantages toward their utilization as permanent graft. The present study has investigated a way to correct these drawbacks by increasing the collagen concentration in controlled conditions. Concentrated collagen hydrogels (CCH) at 1.5, 3 and 5 mg/ml were obtained. The effect of raised collagen concentration on contraction, cell growth and remodeling activities was evaluated for 21 days in culture. Subsequently, in vivo integration of CCH and normal collagen hydrogels (NCH) was assessed. Compared to NCH, CCH contraction was delayed and smaller. At day 21, surface area of CCH at 3 mg/ml was 18 times more important than that of NCH. Whatever the initial fibroblast density, CCH favored cell growth that reached about 10 times the initial cell number at day 21; cell proliferation was inhibited in NCH. Gelatinase A activities appeared lower in CCH than within NCH. In vivo studies in rats revealed a complete hydrolysis of NCH 15 days after implantation. In contrast, CCH at 3 mg/ml was still present after 30 days. Moreover, CCH showed cell colonization, neovascularization and no severe inflammatory response. Our results demonstrate that concentrated collagen hydrogels can be considered as new candidates for dermal substitution because they are is easy to handle, do not contract drastically, favor cell growth, and can be quickly integrated in vivo. Ó 2009 Elsevier Ltd. All rights reserved. 1. Introduction Skin substitutes are among the first examples of tissue engi- neered devices. They are used as permanent grafts for treatment of burns or as temporary dressing for chronic wounds and foot ulcers [1–3]. The challenge to perform a good skin substitute is to find the right balance between restoring basic function of the skin and creating an environment to regenerate the tissue. These bioma- terials are often composed of two layers: an epidermal layer and a dermal layer. The epidermal layer, composed of sheets of autol- ogous keratinocytes, is commercialized under the brand name Epicel Ò [1]. The dermal scaffold is produced with artificial or natural polymers and can include cells [2–5]. The disadvantages of artificial scaffolds are the lack of cell recognition and sometimes the low biocompatibility [6]. Therefore, these biomaterials have to be functionalized and sometimes removed from host organism after healing [4]. Biopolymers such as collagen or fibronectin have numerous advantages in tissue engineering such as low toxicity, low immunogenicity and biodegradability [6–8]. The first scaffold used as dermal substitute was an acellular sponge of collagen and proteoglycans crosslinked with glutaraldehyde and commercial- ized under the brand name Integra Ò [3,9]. To colonize this scaffold, cells have to migrate into large pores. The contacts between Inte- gra structure and cells are in two dimensions and can be consid- ered as non physiological [10]. More recently, another acellular scaffold, a dense collagen matrix concentrated at 40 mg/ml has been developed with interesting mechanical properties [11–13]. In vitro, human dermal fibroblasts migrate into the dense collagen network to reach a distance of about 350 mm after 28 days. Several studies have shown the advantages to include fibro- blasts during dermal substitute fabrication [14,15]. Dermal fibro- blasts secrete extra-cellular matrix macromolecules, soluble factors that diffuse to the overlying epidermis and cytokines which have a role in neovascularization [16]. Fibroblasts have a key role in wound healing and the environment properties determine their behavior [14]. A system in which physiological 3D contacts exist between fibroblasts and the scaffold is the normal collagen hydrogel developed by Bell and co-workers [17]. Collagen hydrogel combined with keratinocyte sheets are used as skin substitutes for treatment of chronic or acute wound under the name Apligraf Ò * Corresponding author. Tel./fax: þ33 1 44 27 65 64. E-mail address: chelary@snv.jussieu.fr (C. Helary). Contents lists available at ScienceDirect Biomaterials journal homepage: www.elsevier.com/locate/biomaterials ARTICLE IN PRESS 0142-9612/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2009.09.073 Biomaterials xxx (2009) 1–10 Please cite this article in press as: Helary C, et al., Concentrated collagen hydrogels as dermal substitutes, Biomaterials (2009), doi:10.1016/ j.biomaterials.2009.09.073